Bacteriophage therapy has the potential to provide an effective method to specifically control the multiplication of various strains of bacteria. However, to be commercially viable, the bacteriophages themselves must show a certain degree of stability to allow for storage.
Various methods have been used to store phage, including freezing at low temperatures, lyophilising, and storing in liquid medium. All methods have shown varying degrees of success at maintaining a high titer of viable bacteriophages.
Prouty (1953, Appl Microbiol, 1:250-351) reported that dessicated bacteriophage of lactic acid producing Streptococci remained viable at 0° C. for 42 months, at 37° C. for 72 months and at 12° C. and 25° C. for at least 78 months. However, there is no mention of the effect of storing desiccated bacteriophage on the titer of the bacteriophage.
Keogh and Pettingill (1966, Appl Microbiol, 14:4421-424) show that bacteriophages for lactic acid producing Streptococci in the presence of whey protein are resistant to freezing and cold storage. Phage stored at 4° C. and −18° C. showed little reduction in the bacteriophage titer; freeze-thaw cycles also showed no significant loss of titer. Warren and Hatch (1969, Appl Microbiol, 17:256-261) report a significant decrease in the titer and viability of a bacteriophage suspension stored (without stabilizers) at 4° C., while storage at −20° C. and 20° C. resulted in the greatest survival of phage. They also report that long term storage of bacteriophages at −20° C. tends to result in the formation of clumps.
Jepson and March (2004, Vaccine, 22:2413-2419) disclose that a liquid suspension of bacteriophages (in either SM buffer or a 1/200 dilution of SM buffer in water) was stable for 6 months at 4° C. and −70° C., with the phage remaining unaffected by freeze-thawing. Increased temperature, between 20° C. and 42° C., resulted in a significant loss of titre. Lyophilisation and immediate reconstitution of bacteriophages in the presence or absence of stabilizers resulted in a loss of titre of 80-95%. Of the bacteriophages remaining following lyophilization in the presence of dry skim milk powder, storage at temperatures between 20° C. and 42° C. resulted in a loss of titre similar to that of the liquid suspension. However, lyophilization in the presence of trehalose resulted in an increase in half-life of bacteriophage between 20° C. and 42° C. The effect of pH of the storage medium was also examined. There was no change in bacteriophage titer over a 24 hour period at pH 3-11. However, the titer dropped rapidly when stored for 5 minutes at pH values below 2.4.
Scott et al (WO 03/093462) discloses the stabilization and immobilization of viruses, including bacteriophage, by covalently bonding the virus to a substrate. This process requires chemicals to activate the substrate and coupling agents to aid in formation of covalent bonds between the substrate and the virus. The use of these reagents increases the expense required for immobilization. Furthermore, the virus or bacteriophage are note able to release to the environment due to their covalent attachment to the substrate. Therefore, the immobilized phage may only act in discreet locations.
Freezing or lyophilisation of bacteriophage suspensions, or bacteriophage suspensions optionally containing stabilizers, are inconvenient methods that require specialized equipment and add to the cost of a commercial preparation. While it is desirable to be able to store bacteriophages in a desiccated state, the process of lyophilization results in a significant loss of titre. Furthermore, the covalent attachment of bacteriophages to a substrate does not allow for the release of the bacteriophages from the substrate and limit its usefulness for our applications. Alternative methods for bacteriophage stabilization are required.